HAMMER: Hairpin-based APOBEC3A-mediated mRNA editing reporter

Area of Science:

• Biochemistry and Molecular Biology
• Genetics and Genomics
• Virology and Cancer Research

Background:

• APOBEC3A enzyme catalyzes cytosine-to-uracil deamination in DNA and RNA.
• Aberrant APOBEC3A activity is linked to cancer-driving mutations and innate immunity.
• The role of APOBEC3A-mediated RNA editing in virus and cancer evolution requires further investigation.

Purpose of the Study:

• To develop a rapid, luminescence-based cellular assay for quantifying APOBEC3A RNA editing activity.
• To establish a tool for characterizing inhibitors of APOBEC3A.
• To investigate the impact of APOBEC3A on herpesviral ribonucleotide reductase activity.

Main Methods:

• Development of the HAMMER (hairpin-based APOBEC3A-mediated mRNA editing reporter) assay.
• Utilizing tandem renilla and firefly luciferase reporters separated by an APOBEC3A substrate sequence.
• Measuring APOBEC3A activity via the ratio of firefly to renilla luciferase luminescence.
• Testing HAMMER specificity and dose-responsiveness with human APOBEC3A and herpesviral constructs.

Main Results:

• HAMMER accurately reports APOBEC3A activity as a reduction in firefly to renilla luciferase ratio.
• The assay is dose-responsive, dependent on catalytic activity, and specific to human APOBEC3A.
• Inhibition of APOBEC3A led to a dose-responsive recovery of firefly luciferase expression.

Conclusions:

• HAMMER is a scalable, user-friendly method for quantifying cellular APOBEC3A RNA editing.
• The assay facilitates the characterization of potential APOBEC3A inhibitors.
• HAMMER provides insights into APOBEC3A's role in biological processes and disease.